The present application is concerned generally with apparatus and methods for the analysis of genes and gene expression; and is particularly directed to the construction and use of a fiber optic biosensor able to detect selectively one or multiple nucleic acid oligonucleotide fragments concurrently.
In less than twenty years, the field of molecular genetics, including the specialty of genetic engineering, has revolutionized the science of biology as a whole and is in the process of restructuring medicine in both diagnostic and therapeutic applications. Not only are individual genes now being isolated and characterized, but also extensive research studies as to how genes function and are regulated in-vivo are being actively pursued. Moreover, many techniques for manipulating and modifying genes have been reported and are today becoming widespread in use and diverse in application. Merely exemplifying the many authorative texts and published articles presently available in the literature regarding genes, gene manipulation and genetic analysis are the following: Gene Probes for Bacteria (Macario and De Macario, editors) Academic Press Inc. 1990; Genetic Analysis, Principles Scope and Objectives by John R. S. Fincham, Blackwell Science Ltd., 1994; Recombinant DNA Methodology II (Ray Wu, editor), Academic Press, 1995; Molecular Cloning. A Laboratory Manual (Maniatis, Fritsch, and Sambrook, editors), Cold Spring Harbor Laboratory, 1982; PCR (Polymerase Chain Reaction). (Newton and Graham, editors), Bios Scientific Publishers, 1994: and the many references individually cited within each of these publications.
Among the many innovative ideas and novel techniques generated by molecular genetic research studies has been the generation of nucleic acid probes for identifying the existence of specific genes, the products of gene expression, and the presence of mutations in one or more genes. By definition, a nucleic acid probe is a DNA or RNA oligonucleotide fragment or peptide nucleic acid (PNA) of known base sequence. Existing as a single-stranded segment of base codons, a nucleic acid probe which will bind to a complementary base sequence of nucleic acids which is the analyte of interest for any purpose. Thus, the oligonucleotide probe, via its selective binding capability, is employed to detect and identify individual gene fragments or nucleic acid sequences present in viruses, bacteria, and other cells serving as samples for scientific, research or medical interest.
In general, any DNA, RNA, or PNA sequential fragment (obtained from any source and regardless of whether the sequence is naturally occurring or synthetically prepared) must meet two essential criteria in order to be truly useful as an oligonucleotide probe. First, the oligonucleotide probe sequence must be as specific as possible for the intended complementary target sequence; and, preferably, bind exclusively with only the complementary target sequence with little or no cross-reaction. Secondly, the oligonucleotide probe must be able to distinguish among closely related nucleic acid base sequences having a substantial degree of homology as well as be able to bind selectively with varying types and sources of nucleic acid fragments having the complementary target sequence as part of its composition. Thus, the size or length of the oligonucleotide probe and the repetitive nature of or copy number for the complementary target sequence will meaningfully affect not only the specificity, but the sensitivity of the probe for detection purposes.
The technique employing an oligonucleotide probe for selective binding to a complementary target sequence is generally termed xe2x80x9chybridizationxe2x80x9d. However, the development of hybridization based assays for the identification of specific genes and gene expression products has been severely limited to date because of major difficulties in: (a) isolating highly specific nucleic acid sequences for use as oligonucleotide probes: (b) developing assay formats that are sufficiently rapid and simple in order to identify even one complementary target sequence in a fluid mixture containing many varieties of different single-stranded oligonucleotides in admixture: and (c) devising non-radioactive detection systems that provide a desired level of sensitivity. Thus several types of DNA (or RNA, or PNA) hybridization assay formats have come into prevalent use.
Four hybridization assay formats are commonly employed today. Each of these hybridization detection formats suffers from relatively poor sensitivity, although various target sequence amplification techniques (such as PCR) have also been developed to reduce the severity of this problem. The four most commonly used types of hybridization assay formats are: the Southern blot technique; the dot or spot blot technique; in-situ hybridization; and sandwich hybridization assays. As with the selection of an appropriate oligonucleotide probe, the choice of a hybridization assay format often rests upon the degree of specificity and sensitivity that is required for the particular analysis; and upon the factors of speed, reliability, and ease of performance and interpretation of the assay resultxe2x80x94which varies markedly among the different assay formats.
In Southern blot assays, specimen DNA is isolated and purified prior to restriction endonuclease digestion; followed by separation of the digestion products by electrophoresis on an agarose gel, denaturation of the DNA in the gel, and transfer of the denatured DNA fragments to a solid matrix such as a nitrocellulose membrane. The DNA bound to the solid matrix is then hybridized in the presence of radioactively labeled DNA targets to establish homology between the probe and target DNA. Hybridization of the targets to the probes is detected by autoradiography and often requires several days or weeks of exposure. This format is thus often too lengthy and cumbersome for routine or large-scale analyses of many specimens.
The dot-blot procedure also requires that specimen DNA be isolated and purified before being denatured and applied to a suitable solid matrix (such as nitrocellulose). Hybridization to the matrix-bound DNA is then performed using probe-specific targets. The hybridization of target DNA to the probe DNA is detected either by autoradiography or by visual inspection using non-radioactive detection procedures. The spot-blot assay format is similar except that specimens or specimen lysates are directly applied to the solid matrix without prior extraction of their DNA. Although this assay format allows many different samples to be processed at one time, these assays are often limited to high background noise that complicates the interpretation of results and is also subject to lengthy time of processing for each sample to be evaluated.
The in-situ hybridization technique intends that the DNA or RNA in the cells of a fixed tissue section or fixed culture cell be hybridized to DNA probes directly on a microscope slide. The results are determined by microscopy if non-radioactive detection systems are used and by autoradiography if radioisotopes are employed for the targets. This assay format can detect the presence of only a few copies of the target DNA sequence to be hybridized. This conventional in-situ hybridization assay is not suitable for screening large numbers of specimens due to the need to separate and remove extraneous cellular materials from the sample prior to addition of the labeled target.
Lastly, the sandwich hybridization assay requires that at least two different specific probes hybridize to the target DNA of interest, rather than just one probe alone. In this format, the first probe (the capture sequence) is bound to a solid support and is allowed to bind (capture) the specimen DNA. A second probe (the signaling probe) with a sequence that is adjacent or close to the capture sequence on the target DNA molecule is then allowed to hybridize to the support-bound target DNA. This signaling probe can be labeled with either radioactive or non-radioactive labels and the removal of non-specific cellular material in the first step of the procedure enhances the specificity of the hybridization assay by reducing the effects of contaminating tissue or debris.
More recently however, the value of using immobilized spatially distinguishable, hybridization probes for concurrent analyses of multiple gene sequences has been recognized and resulted in the development of miniaturized hybridization assays using solid matrix assays [Southern, E. M., Trends in Genetics 12: 110-115 (1956)]. Thus, hybridization using said matrix arrays have been performed on glass surfaces [Maskos, U. and E. M. Southern, Nuc. Acids. Res. 20: 1679-1684 (1992); Guo et al., Nuc. Acid. Res. 22: 5456-5465 (1994)]; on microtiter plates [Kalakowski et al., Anal. Chem. 68:1197-1200 (1996); Nikiforov et al., Nuc. Acids Res. 22: 4167-4175 (1994); Rasumussen et al., Anal. Biochem. 198: 138-142 (1991)]; on plastic sheets [Matson et. al., Anal. Biochem. 224: 110-116 (1995)]; on thin polymer gels [Khrapko et al., J. DNA Seq. Map 1:375-388 (1991)]: and using semiconductor devices [Eggers et al, Bio Techniques 17: 516-524 (1994); Kreiner, T, Am. Lab.: 39-43 (1996)]. In addition, the desire for using non-radioactive means for detection have caused a surge of interest in means for detection of hybridization on solid matrix supports which employ fluorescence [Kumke et. al., Anal. Chem. 67: 3945-3951 (1995); Piunno et. al. Anal. Chim. Acta. 288: 205-214 (1994)]; chemiluminescence [Ito et. al. J. Neurosci. Methods 59: 265-271 (1995); Nguyen et. al., Biosen. Bioelectron. 7: 487-493 (1995)] evanescent wave technology [Graham et. al., Biosen. Bioelectron. 7: 487-493 (1992): Strachan et. al Lett. Aor:. Microbiol. 21:5-9 (1995). Watts et. alAnal. Chem. 67: 4283-4289 (1995)]: confocal microscopy [Fodor et. al. Nature (London) 364: 555-556 (1993)]: light scattering [Stimpson et. al., Proc. Natl. Acad. Sci. USA 92: 6379-6383 (1995)]: electrochemistry [Millard et. alAnal. Chem. 66: 2943-2948 (1994): Pandey et. al. Anal. Chem. 66:1236-1241 (1954): Hashimoto et. al., Anal. Chim. Acta. 286:219-224 (1994)]: and surface resonance phenomena [Yamaguchi et. al, Anal. Chem. 65: 1925-1927 (1993)].
Despite these recent innovations using probes immobilized on solid matrix arrays the major obstacles and limitations of hybridization methods generally continue to restrict and contain the currently available techniques and formats. These demands and limitations include a requirement for a large sample volume; an inability to perform multiple analyses concurrently in real time; a requirement for a relatively high concentration of target DNA (the complementary target sequence) in the fluid sample; an inability to detect multiple species concurrently; relatively slow kinetics for hybridization to occur between the target sequences and the immobilized probes within the assay format; and a dependence upon lengthy assays. Moreover, despite the use of new in-vitro amplification techniques such as the polymerase chain reaction procedure, the problems of assay sensitivity, lengthy times for analysis, the quantum of background signal noise, and the inability to detect more than one target nucleic acid sequence at a time remain as recurring handicaps and continuing obstacles for each of these techniques. It will be recognized and appreciated by persons working in this field today, therefore, that the development of a unique biosensor which overcomes and eliminates most, if not all of these major limitations and procedural hindrances would be seen as a major advance and unforeseen improvement in this art.
The present invention provides optical sensors for detecting nucleic acids in a fluid sample. The sensors comprise a preformed, unitary fiber optic array comprising a plurality of optical fiber strands, the strands being disposed co-axially and joined along their lengths. The fiber optic array has a proximal and distal end, and each of the ends are formed by multiple strand faces of the strands. At least one of the array ends presentes a discrete fiber optic array surface for introduction and conveyance of light energy. The array further comprises at least a first and a second nucleic acid attached to a first and second portion, respectively, of the distal array end. The first nucleic acid is optically coupled to and in optical communication with a first of the multiple end faces at the distal array end, and the second nucleic acid is optically coupled to and in optical communication with a second of the multiple end faces at the distal array end.
In an additional aspect, the present invention provides methods of making a fiber optic sensor. The method comprises providing a preformed, unitary fiber optic array as outlined above, and contacting at least a first nucleic acid and a photoactivatable compound with at least a first distall optic fiber strand end face, such that the first nucleic acid is attached to the end face by introducing light energy to the first optic fiber strand.
In a further aspect, the invention provides methods of detecting at least one target sequence in a fluid sample. The method comprises providing a preformed, unitary fiber optic array as outlined herein, and contacting the distal end of the array with the sample. The presence or absence of the target sequence is then detected.